ATP plays important roles in the sensory and motor functions of the urinary bladder. ATP released from parasympathetic fibers can excite the detrusor muscle, and ATP released from the urothelium in response to bladder distension can indirectly modulate detrusor contractility by activating afferent fibers, stimulating suburothelial myofibroblasts and inducing release of other signaling molecules from the urothelium. Pathological conditions can increase the purinergic component of the neurogenic detrusor contraction and also augment urothelial ATP release. In diabetes various urodynamic abnormalities can develop, including increased bladder activity. We have proposed that increased sensitivity of diabetic bladders to purinergic stimulation was related to upregulation of specific purinergic receptor (P2R) subtypes in the detrusor muscle, and that amplification of ATP signaling would directly contribute to the development of bladder overactivity in diabetes. Based on recent findings we have now evidence that ATP signaling in also amplified in the diabetic bladder urothelium. Expression of P2Rs, particularly the P2X7R and P2X3R subtypes, is markedly increased in the urothelium of STZ-diabetic rat bladders and responses to ATP are higher in STZ-diabetic urothelial cells. These findings combined with demonstrations that P2X7R activation can induce release of both ATP and prostaglandin (PGE2), suggest that not only the sensitivity to ATP but also ATP and PGE2 release from urothelial cells is increased in diabetic bladders. In this context, afferent signaling from the bladder as well as ability of urothelial cells, suburothelial myofibroblast and smooth muscle cells to communicate would be significantly enhanced and likely contribute to increase bladder activity in diabetes. To test the hypotheses that diabetes increases urothelial ATP signaling and communication between bladder compartments, and that enhanced ATP signaling within and from the bladder contributes to the development of bladder dysfunction in diabetes we will use a combination of molecular biology, biochemistry, pharmacology, in vitro subcellular imaging and whole animal physiology approaches. These studies are expected to lead to novel understanding of the interplay among specific membrane receptors and channel proteins involved in intercellular signaling between urothelium and bladder smooth muscle, and reveal novel therapeutic targets and strategies to ameliorate the symptoms of bladder dysfunction in diabetes. PUBLIC HEALTH RELEVANCE: The studies proposed in this RO1 application are expected to further our understanding of the interactions between the bladder urothelial, suburothelial and smooth muscle compartments and of how changes in the expression of main components of the urothelial ATP signaling contribute to the development of bladder dysfunction in diabetes.